Evaluation and Validation of a Real-Time Polymerase Chain Reaction Assay for Rapid Identification of Bacillus anthracis Supplement

During the recent outbreak of bioterrorism-associated anthrax in the United States, 11 patients were diagnosed with inhalational anthrax and 7 with cutaneous anthrax (Table 1 and 2) (1–6). During the extensive epidemiologic investigation, >125,000 clinical and environmental specimens were collected and analyzed for Bacillus anthracis, the causative agent of anthrax. We used the Laboratory Response Network (LRN) polymerase chain reaction (PCR) assay (real-time PCR assay) during the anthrax outbreak to detect B. anthracis DNA in environmental samples and clinical specimens. This assay provided 100% sensitivity and specificity when evaluated and validated on our panel of diverse bacterial isolates. On clinical specimens, this assay was one of three used to confirm anthrax cases when isolation of B. anthracis failed after antimicrobial drug treatment was initiated. In these culture-negative cases, laboratory confirmation was based on at least two supportive laboratory tests including this PCR, immunohistochemical stain (IHC), or anti-protective antigen (PA) titer (immunoglobulin [Ig]G enzyme-linked immunosorbent assay [ELISA]). PCR assays have not been previously used in an outbreak setting to detect B. anthracis directly in clinical specimens in a real-time manner. We evaluated the use of this assay on the clinical specimens and environmental samples received during the outbreak.

from each specimen. From one aliquot DNA was extracted with a MagNa Pure LC instrument (Roche Diagnostics GmbH, Mannheim, Germany) by employing a DNA isolation kit I with the "High Performance" protocol. In addition, select specimens were extracted in duplicate with a Qiagen DNA Mini Kit (Qiagen, Valencia, CA) per manufacturer's instructions. A second aliquot was used to inoculate bacteriological media for isolation of B. anthracis (7).
Specimens from patients meeting the definition for confirmed anthrax and from those in whom the diagnosis was excluded were tested by LRN PCR assay and traditional culture using the methods described above. For specimens that were unavailable for testing at Centers for Disease Control and Prevention (CDC), culture results reported by the clinical laboratories of the patient's treating facility were used for case confirmation.
The performance of the LRN PCR assay was compared to that of traditional culture methods by testing environmental specimens collected from throughout the United States during the course of the outbreak by both methods. B. anthracis spores were eluted from swab specimens and other environmental samples in 2.5% pluronic F-68 (Sigma, St. Louis, MO) and then collected by centrifugation through an Ultrafree-CL, 0.45 uM, PVDF membrane filter (Millipore, Bedford, MA). Spores were eluted from the filters with 2.5% pluronic F-68, used to inoculate bacteriologic media, and added directly to real-time PCR assays without further purification or DNA extraction.
Two hundred seventy-nine clinical specimens were tested by both culture and real-time PCR: 92 were from 9 patients with inhalational anthrax, 33 from 7 patients with cutaneous anthrax, 12 from 4 patients with suspect cutaneous anthrax, and the remaining 142 from 74 patients in whom anthrax was excluded (Table 3). Of the 92 specimens from the inhalational anthrax cases, 5 (all blood specimens) were positive by both methods. Of the remaining 87, all were culture negative, but 29 (33%) were positive by the PCR assay. These included serum, sputum, pleural fluid, and tissue specimens (Table 3). Of the 33 specimens from the cutaneous anthrax cases, none were culture positive, but positive PCR results were obtained on a single blood specimen and two skin biopsy specimens. None of the 142 specimens from 74 patients without anthrax had positive results on culture or PCR. including 9 inhalational anthrax patients and 5 cutaneous patients (2 were confirmed by culture, 3 by IHC and serology, and 2 by IHC and PCR). PCR was also performed on specimens from 74 patients in whom anthrax was excluded.
Culture was performed on specimens from 13 anthrax patients in whom the diagnosis could be confirmed using non-culture methods, including 8 patients with inhalational anthrax and 5 patients with cutaneous anthrax. Culture was also performed on specimens from 74 patients in whom anthrax was excluded.

Inhalational Cases
Of the 11 patients with inhalational anthrax, 8 had blood cultures performed before the initiation of antimicrobial drug therapy, and cultures were positive in all eight at the hospital where patients were initially treated. At CDC, B. anthracis was also isolated from blood cultures of patient 5 (two blood cultures collected immediately before the start of the antimicrobial drug therapy), patient 6 (one blood culture collected on the same day antimicrobial drug therapy started),and patient 11 (two blood cultures collected the day before antimicrobial drug therapy). In contrast, 44 blood specimens were cultured from five patients (patients 2, 8, 9, 10, 11) ( all four of these specimens were PCR positive ( Figure).
Seven postmortem tissue specimens were collected from three patients. Samples from a lymph node and lung tissue from one patient (patient 10) and a lymph node sample from another patient (patient 11) were PCR positive. All others were negative (Table 4).
Two sputum samples were tested. A sputum sample from patient 2 was received 5 days after the administration of antimicrobial drugs, and it was PCR negative. The second sputum was obtained on day 2 after the administration of antimicrobial drugs and was PCR positive (patient 11).
Of the seven patients with cutaneous anthrax, two had blood cultures performed before administration of antimicrobial drug therapy at the medical facility where patients were treated, and one patient had a positive result ( Table 2,  PCR was positive on one fixed tissue sample (patient 2), obtained 14 days after onset date, and on a fresh frozen tissue (patient 6) received 6 days after antimicrobial drugs were administered. In addition, one frozen tissue sample was received from a single patient before antimicrobial drug therapy; both culture and PCR were negative.Four additional cutaneous cases were defined as suspect because only one supportive laboratory test was positive; for three of the cases, serologic testing was positive, and for the fourth, IHC of an arm biopsy specimen was positive. A total of 12 specimens (7 blood specimens, 3 sera, 2 swabs) collected from these four patients were tested by PCR and culture; all were PCR and culture negative.

Patients without Anthrax
One hundred eighty-six clinical specimens were collected from 74 patients who were subsequently determined not to have either inhalational or cutaneous anthrax; 142 specimens were culture and PCR negative (PCR specificity of 100%, 95% confidence interval 99% to 100%), and the remaining 44 tested by PCR only were also negative.

Real-Time PCR in Environmental Specimens
One hundred forty environmental specimens were analyzed by both culture and Overall, B. anthracis was isolated from 8 (73%) of 11 patients with confirmed inhalational anthrax while the LRN PCR was positive for 8 (89%) of 9 patients tested.
One case in which only two blood cultures were tested yielded negative results for both culture and PCR. Of the seven patients with confirmed cutaneous anthrax, B. anthracis was isolated from two patients (29%), and the LRN PCR was positive for three (43%). Evaluation of the LRN PCR and its performance on clinical specimens was not conducted as a true prospective study as we were, to a degree, limited by the number and type of specimens available, as well as by the emergent response needed to establish the microbiologic diagnosis. However, the number and variety of clinical samples were substantial enough to allow statistically significant comparisons with the current standard, culture. Also, the fact that laboratory confirmation was obtained by either and vaccines were prevented when B. anthracis was rapidly excluded from differential diagnosis.

Figure.
Results of polymerase chain reaction testing of clinical specimens (for which dates of collection were available) from a patient with inhalational anthrax (patient 2), are illustrated by date of collection relative to the initiation of antimicrobial drug therapy. Bacillus anthracis was not recovered from any of these specimens on which culture was attempted (data not shown). A.